S150: BIOLOGY AND FUNCTION OF CIRCULAR PCMDT1 IN CHRONIC MYELOID LEUKEMIA IN BLAST CRISIS (CML-BC)
Authors: A. Prathap Urs, D. Papaioannou, R. Buisson, S. Khanal, M. Karunasiri, S. Kauppinen, A. Dorrance, R. Garzon
Journal: HemaSphere (2022)

Background: The prognostic and biologic significance of circular RNAs (circRNAs) in patients with acute myeloid leukemia (AML) has been reported. Circular PCMTD1 (cPCMTD1) was among the circRNAs that were prognostic in this disease. Aims: To study the functional role of cPCMTD1 in leukemias. Methods: We used RNase H-recruiting, locked nucleic acid-modified oligonucleotides (gapmers) for knock-down (KD) experiments. Cell cycle analyses were performed with bromodeoxyuridine and 7-actinomycin D staining. Chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU) were used for DNA fiber assays. Results: To study whether cPCMTD1 is important for leukemia, we KD-cPCMTD1 using gapmers that specifically degraded the circular transcript without affecting the levels of the linear PCMTD1. Among the 8 AML cell lines that were tested, we found that depletion of the cPCMTD1 led to a decrease in the proliferating fraction and potent G2/M blockade of only K-562 and LAMA-84 CML-BC cells. In contrast, linear PCMTD1-KD did not have any effect on cell cycle. Mass cytometry (CyTOF) experiments validated the cell cycle blockade after cPCMTD1-KD and identified an increase of H2AX phosphorylation. We validated this finding with western blots and could also show that cPCMDT1-KD led to an increase in the phosphorylation of the ATM, ATR, CHK1, DNA-PK and RPA32 proteins. Further, DNA fiber assays detected a global decrease in the length of CldU- and IdU-labeled fibers as well as of the IdU/CldU ratio in the cPCMTD1-KD cells compared to controls. These results were confirmed by COMET assays. Taken together these data indicate that cPCMTD1-KD causes impaired DNA replication in CML-BC. Then, we examined whether we found in CML-BC cell lines was also true for patients. Thus, we measured cPCMDT1 expression in a panel of CML patients in chronic phase (n=15), accelerated phase (n=4) and blast crises (n=7) and we found a significant increase of cPCDMT1 in CML-BC with respect to chronic and accelerated phases. Next, we performed cPCMDT1-KD in CML-BC patient samples and found increase of H2AX phosphorylation. Like the cell line data, cPCMDT1-KD in 3 samples from AML patients had no functional effect. Finally, after confirming that cPCMDT1 is most abundant in the cytoplasm, in silico analysis and polysome profiling experiments indicated that cPCMDT1 encodes for a small protein. We generated a custom antibody that detected the predicted protein of 30kD, which was depleted after cPCMDT1-KD. Immunoprecipitation experiments followed by mass spectrometry analysis using our custom antibody showed a strong and specific enrichment of the BTR complex (BLM, TOP3A and RMI1), which is implicated in DNA replication and repair. cPCMDT1-KD had no effect on the total amount of each of the three proteins but reduced their interaction and the amount of the formed BTR complex. To validate the therapeutic potential of this strategy we developed a CML-BC PDX model by transplanting a CML-BC patient sample into NSGS mice. This PDX is very aggressive, and mice die from disease by 20 weeks. We are currently treating these mice in vivo with lipid nanoparticle anti-cPCMDT1 gapmers. Results will be updated in the meeting. Summary/Conclusion:cPCMDT1 is a circRNA with protein-coding potential that is over-expressed in CML-BC. cPCDMT1 is critical for the proliferation of CML-BC through the regulation of DNA replication and may represent a novel target for therapy.